Innumerable qualitative and quantitative tests are available for detecting the presence or level of particular substances in a sample. Sources of such samples range from industrial environments such as mines, wastewater processing, food quality, soil testing, among many others. In the medical field, tests for substances in bodily fluids are well known, and are aids to prognostication, diagnosis, and monitoring the progression and treatment of various conditions and diseases. In many cases, multiple tests are performed on a sample and a health care professional then makes a presumptive diagnosis based on the various levels of particular analytes in the sample, among other information gained, for example, from examining a patient.
In certain circumstances, particular in the emergency room and ambulance call, time is of the essence in arriving at a diagnosis and initiating appropriate therapy to intervene in the morbidity and mortality of a rapidly deteriorating condition. One such example is diagnosis of a heart attack in an individual with chest pain or recent onset. Multiple diagnoses may be attributable to chest pain, yet diagnosis based on electrocardiogram or levels of cardiac markers released into the circulation are needed for a confirmatory diagnosis and initiation of a course of therapy, which would be unwise in a patient not having a heart attack. Thus, the need for rapid and accurate, early diagnostic tests is apparent for such emergency conditions.
Although such early tests are available, even such tests are not without flaws. For example, diagnosis of a heat attack within six hours of the onset of chest pain is difficult to perform with a single test. While the cardiac marker troponin I has been recently adopted as a single and highly accurate indicator, it is not detectable until after about six hours, leaving a large window where early initiation of treatment would be highly desirable but dangerous without an accurate diagnosis. Another cardiac marker, myoglobin, is released into the circulation earlier than troponin I, but is not specific for cardiac tissue, as skeletal muscle damage also releases myoglobin into the circulation. Additional tests may be performed together with myoglobin to attempt to identify its origin, in order to improve the accuracy of an early diagnosis.
The foregoing example of heart attack is merely one example of a myriad of diagnoses, which if to be carried out with a high degree of accuracy, need additional, corroborative tests. Although the combination of multiple assays performed simultaneously increases diagnostic precision, it is undesirable in that it also increases the complexity of the testing, the coordination of the timing of the separate test procedures and availability of the information, and the amount of information that must be processed manually or otherwise, often under emergency conditions.
It is towards the simplification of multiple analyte diagnostic tests to provide a single readout reliably indicative of a particular diagnosis that the present invention is directed.
In its broadest aspect, the present invention provides an assay useful to determine the relative levels at which different analytes are present in a sample. In the present assay, the extent of the readout is related to the level of the first analyte and that of the second analyte. Thus, in one embodiment, the readout provides either a ratio of the level of the first analyte to the second, or the difference in levels between the first analyte and the second analyte.
The assay of the invention is useful for situations in which a ratio or difference between the levels of the first and second analytes is diagnostically useful, and a single readout that takes the two values into consideration in generating a single differential value can be as informative and directive of further action as would be obtaining the individual values and mentally evaluating or arithmetically calculating the difference or ratio, and then acting upon the result. The method of the invention simplifies decision making by internally integrating the results of at least two individual analyte levels.
By way of non-limiting example, the first analyte and second analytes may be markers useful for determining the health status of an individual, wherein the ratio or difference among the markers is diagnostically informative. In a particular embodiment, elevated levels of the first analyte may be indicative of a life-threatening medical event only if the level of the second analyte is not elevated. In another embodiment, the second analyte also being elevated is indicative of an event. In a third and preferred embodiment, an analyte may originate from different bodily sources and the origin is diagnostically useful; the assay of the invention is useful for identifying the source of elevated levels of the first analyte. In this embodiment, the format of the assays of the invention takes advantage of the co-release of another analyte from the bodily source other than the intended source (referred to herein as a non-target-source marker) whose level is effectively subtracted from that of the total level of desired analyte to provide in a single test, a readout specific to the analyte source.
In one broad aspect, the single assay for a preselected analyte is indicated as the level of the first analyte reduced proportionally by the level of a second analyte present in the sample. A reading is obtained only if the first analyte is present, and the detected level of the first analyte is reduced as the level of the second analyte increases.
In the present assay, the relative presence or level of first and second analytes in a given sample is revealed by utilizing a labeling reagent for one of the two analytes that labels that analyte through a reaction that is inhibited by the other analyte. More particularly, in the present assay, the readout is dependent on binding, to one of the analytes selectively, of a labeling reagent complex the formation of which is inhibited by any second analyte present in the sample. Thus, labeling of the analyte targeted by the labeling reagent proceeds in the absence of the second analyte, and a reading is obtained. No reading is obtained when the first analyte is absent from the sample. When both analytes are present in the sample, the labeling reaction proceeds but in a manner that is competitively inhibited by the second analyte. Thus, the relative levels at which the first and second analytes are present in the sample is reflected by the extent to which the first analyte is labeled, and this is ultimately reflected in the readout obtained following performance of the assay.
Thus, in one of its aspects, the present invention provides a method useful to assay a sample to detect the presence or relative levels therein of first and second analytes, the method comprising the step of bringing the sample into contact with a labeling reagent means adapted to form a labeling complex that binds to and thereby labels the first analyte, wherein formation of the labeling complex is inhibited by second analyte present in the sample.
In a preferred aspect of the invention, the labeling reagent means comprises two components: a labeled binding partner for the second analyte, and a conjugate formed by coupling of a second analyte itself and a binding partner for the first analyte. When second analyte is absent, the first analyte is thus labeled by formation of complexes between the first analyte and the first analyte binding partner, and the second analyte and the labeled second analyte binding partner. When present in the sample, however, the second analyte becomes a competitor for binding to the labeled second analyte binding partner, and thereby inhibits binding of that labeled second analyte binding partner to the conjugate, thus reducing labeling of the first analyte.
In a particular embodiment, a method is provided for identifying in a sample the presence or level of a first analyte above the level of a second analyte comprising the steps of
(a) forming a reaction mixture by contacting the sample with reagent means for labeling the first analyte, the labeling reagent means comprising a mobile, labeled binding partner to the second analyte, and a conjugate between the second analyte and a binding partner to the first analyte;
(b) contacting the reaction mixture with an immobilized binding partner to the first analyte;
wherein the extent of formation of a complex comprising the mobile, labeled binding partner to the second analyte, the conjugate between the second analyte and the binding partner to the first analyte, the first analyte, and the immobilized binding partner to the first analyte, is indicative of the presence or level of the first analyte in the sample reduced by the level of the second analyte in the sample. Desirably, the reaction is staged by first bringing the sample into contact with the mobile, labeled binding partner to the second analyte to allow any second analyte in the specimen to become bound thereto, and then presenting the conjugate before finally contacting the resultant mixture with immobilized binding partner to the first analyte. In this way, the inhibitory effect of sample-borne second analyte is maximized, by allow it to react first with the labeled second analyte binding partner before allowing the conjugate to compete therewith for binding.
By way of non-limiting example, the aforementioned binding partners may be antibodies. The label may be colloidal gold. The sample may be, by way of non-limiting example, a bodily fluid, wastewater, a foodstuff; preferably, it is a bodily fluid such as whole blood, serum, plasma, or urine. By way of example, the first analyte may be a cardiac marker, such as myoglobin, and the second analyte may be a different analyte co-released from a non-cardiac source along with the first analyte, such as carbonic anhydrase III which is released from damaged skeletal muscle along with myoglobin. For determining the level of myoglobin originating from the heart, the mobile, labeled binding partner to the second analyte may be a gold-labeled monoclonal anti-carbonic anhydrase III antibody, the conjugate between the second analyte and a binding partner to the first analyte may be a conjugate between carbonic anhydrase III and an anti-myoglobin monoclonal antibody, and the immobilized binding partner to the first analyte may be an anti-myoglobin monoclonal antibody.
The conjugate between the second analyte and a binding partner to the first analyte may be a covalent conjugate between the members, such as is achievable using a homobifunctional or heterobifunctional cross-linking agent or carbodiimide, or it may comprise a single-chain polypeptide on which reside both the second analyte, or an epitope thereof, and a binding partner, or binding portion thereof, to the first analyte, such that each member retains its desired activities within the conjugate or single-chain polypeptide. For example, the conjugate between an antibody to myoglobin and carbonic anhydrase III may include a single-chain polypeptide comprising carbonic anhydrase III and the immunoglobulin heavy chain, which when assembled into the functioning antibody, provides binding sites for myoglobin and a carbonic anhydrase III portion to which the labeled anti-carbonic anhydrase III antibody may bind. The analyte portion of any of the conjugates herein may be the full-length analyte or a fragment bearing the epitope recognized by the corresponding binding partner. The foregoing example may be used to diagnose a heart attack by indicating an elevated level of myoglobin exists over that which may derived from a non-cardiac source. In this case, the level of cardiac and skeletal (i.e., total) myoglobin detected in the assay is reduced by the amount of carbonic anhydrase III present in the sample, the latter equivalent to the level of skeletal muscle-derived myoglobin.
In a second embodiment, a homogeneous assay similar to that above is provided which employs slightly different reagents, but applies the same principles. In this embodiment, a further binding interface is incorporated into the labeling reaction. Particularly, the conjugate between the first analyte binding partner and the second analyte instead introduces a further biotin/streptavidin interaction, and the conjugate thus is represented by two reagents; one in which biotin is conjugated with either the first analyte antibody or the second analyte, and another in which streptavidin or a biotin-binding component thereof is conjugated with the other of the first analyte antibody or the second analyte. In an embodiment, the conjugate reagents are a first conjugate between first analyte antibody and biotin, and a second conjugate between streptavidin and the second analyte.
Thus, a method is provided for identifying in a sample the presence or level of a first analyte above a second analyte, the method comprising conducting an assay following the steps of
(a) forming a reaction mixture by contacting the sample with
(1) a mobile, labeled binding partner to the second analyte,
(2) a conjugate between the second analyte and streptavidin; and
(3) a biotinylated binding partner to the first analyte; and then
(b) contacting the reaction mixture with an immobilized binding partner to the first analyte;
wherein the extent of formation of a complex comprising the mobile, labeled binding partner to the second analyte, the conjugate between the second analyte and streptavidin, the biotinylated binding partner to the first analyte, the analyte, and the immobilized binding partner to the first analyte, is indicative of the presence or level of the first analyte in the sample reduced by the level of the second analyte in the sample.
Desirably, the assay is performed by staging the addition of reagents in step (a), so that sample is first exposed to the mobile labeled binding partner to the second analyte so that any second analyte in the sample becomes bound thereto, before addition of the competitive-binding conjugate between streptavidin and the second analyte.
By way of non-limiting example, the aforementioned binding partners may be antibodies. The label may be colloidal gold. Streptavidin, or a biotin-binding component thereof, or another biotin-binding partner may be used. The sample may be a biological sample such as a bodily fluid: examples include whole blood, serum, plasma, and urine. By way of example, the first analyte may be a cardiac marker, such as myoglobin, and the second analyte may be a different analyte co-released from a non-cardiac source along with the first analyte, such as carbonic anhydrase III released from damaged skeletal muscle along with myoglobin. For determining the level of myoglobin originating from the heart, the mobile, labeled binding partner to the second analyte may be a gold-labeled monoclonal anti-carbonic anhydrase III antibody, the conjugate between the second analyte and a biotin-binding molecule a conjugate between carbonic anhydrase III and streptavidin, a conjugate between biotin and a binding partner to the first analyte may be a biotinylated anti-myoglobin monoclonal antibody, and the immobilized binding partner to the first analyte may be an anti-myoglobin monoclonal antibody.
The conjugate between the second analyte and a binding partner to biotin may be a covalent conjugate between the members, such as is achievable using a homobifunctional or heterobifunctional cross-linking agent or carbodiimide, or it may comprise a single-chain polypeptide on which reside both the second analyte or an epitope thereof, and streptavidin or the biotin-binding portion thereof, such that each member of the conjugate of single-chain polypeptide independently retains its respective binding activity. For example, the conjugate between streptavidin and carbonic anhydrase III may be a single-chain polypeptide comprising carbonic anhydrase III, or an epitope thereof, and streptavidin or a biotin-binding portion thereof, thus providing binding sites for both a biotinylated antibody and anti-carbonic anhydrase III antibody. As mentioned above, the analyte portion of any of the conjugates herein may be the full-length analyte or a fragment bearing the epitope recognized by the binding partner. The foregoing example may be used to diagnose a heart attack by indicating an elevated level of myoglobin exists over that which may derived from a non-cardiac source, in the same manner as described in the previous embodiment. Variations on these embodiments in the selection of the reagents and operation of the components of the test are fully embraced within the spirit and scope of the present invention.
In a preferred embodiment, the first or preselected analyte is an analyte originating from a target source whose level is desirably measured over the same analyte originating from a source other than the target source. A second analyte is a marker that is released from the non-target (other) source in proportion to the level of first analyte released from the non-target source. The assay of the invention subtracts or proportionally reduces, depending on binding partner affinities, from the total level of first analyte (from the target and non-target source) the level of the second analyte, which effectively subtracts the level of the first analyte derived from the non-target source from the readout value.
By selecting the affinities of the binding partners to the analytes and ratio of the components in the conjugates of the invention, as well as using fragments of the analytes comprising the epitope of the analyte recognized by the binding partners, the relative sensitivity of the assay to the first analyte and especially the reduction in value achieved by the presence of any second analyte in the sample may be adjusted to provide an assay which essentially reads out the ratio between the first analyte and the second analyte. For example, if the second analyte is released from the non-target tissue in very small amounts compared to the amount of the target analyte released from the non-target source, use of a higher affinity antibody in the conjugate of the invention to the second analyte in contrast to a lower affinity antibody to the first analyte will increase the sensitivity of the assay to any second analyte present in the sample. Such variations in the invention are within the realm embraced here, and one of skill in the art by following the teachings herein will readily prepare an assay for other analytes or with other operating characteristics, sensitivities, ranges, or other parameters.
Thus, in one embodiment, a method is provided for identifying in a sample the presence or level of a preselected analyte originating from a target source, wherein any level of the preselected analyte in the sample originating from a source other than the target source is associated with an increased level in the sample of a marker from the source other than the target source, the method comprising conducting an assay following the steps of
(a) contacting the sample with a labeling reagent comprising (1) a mobile, labeled binding partner to one of the preselected analyte and the marker, (2) a conjugate between the marker and a binding partner to the preselected analyte; and then
(c) contacting the resulting sample with an immobilized binding partner to the other of the marker and the preselected analyte;
wherein the extent of labeling of said immobilized binding partner is indicative of the presence or level of the preselected analyte in the sample reduced by the level of the marker originating from the source other than the target source.
In a preferred embodiment, the immobilized binding partner is a binding partner for the analyte, and the labeled, mobile binding partner is a binding partner for the marker.
By way of non-limiting example, the aforementioned binding partners may be antibodies. The label may be colloidal gold. The sample may be a biological sample such as whole blood, serum, plasma, or urine. By way of example, the preselected analyte may be a cardiac analyte, such as myoglobin, and the corresponding marker may be carbonic anhydrase III. For determining the level of myoglobin originating from the heart, the mobile, labeled binding partner to said marker may be a gold-labeled monoclonal anti-carbonic anhydrase III antibody, the conjugate between the marker and a binding partner to the preselected analyte may be a conjugate between carbonic anhydrase III and an anti-myoglobin monoclonal antibody, and the immobilized binding partner to the preselected analyte may be an anti-myoglobin monoclonal antibody. The conjugate between the marker and a binding partner to the preselected analyte may be a covalent conjugate between the members, such as is achievable using a homobifunctional or heterobifunctional cross-linking agent or carbodiimide, or it may comprise a single-chain polypeptide on which reside both the marker or an epitope thereof and a binding partner or portion thereof, such that each member retains the desired activities in the conjugate or single-chain polypeptide. For example, the conjugate between an antibody to myoglobin and carbonic anhydrase III may include a single-chain polypeptide comprising carbonic anhydrase III and the immunoglobulin heavy chain, which when assembled into the functioning antibody, provides binding sites for myoglobin and a carbonic anhydrase III portion to which the labeled anti-carbonic anhydrase III antibody may bind. The foregoing example may be used to diagnose a heart attack, as described above.
In a second embodiment, an assay similar to that above is provided which employs variations in the components, but provides the same objectives. Thus, a method is provided for identifying in a sample the presence or level of a preselected analyte originating from a target source, wherein any level of said preselected analyte in the sample originating from a source other than the target source is associated with an level in the sample of a marker from the source other than the target source, the method comprising conducting an assay following the sequential steps of
(a) first contacting the sample with an analyte labeling reagent comprising
(1) a mobile, labeled binding partner to one of the preselected analyte or the marker,
(2) a conjugate between the marker and one of biotin and streptavidin, and
(3) a binding partner to the preselected analyte conjugated to the other of biotin and streptavidin; and then
(b) contacting the sample with an immobilized binding partner to the other of the preselected analyte and the marker;
wherein the extent of labeling of the immobilized binding partner is indicative of the presence or level of the preselected analyte in the sample reduced by the level of the marker in the sample originating from the source other than the target source.
In a preferred embodiment, the analyte labeling reagent comprises (1) a mobile, labeled binding partner to the marker, (2) a conjugate between the marker and streptavidin, and (3) a biotinylated binding partner to the preselected analyte.
In other preferred embodiments, the immobilized binding partner is a binding partner for the analyte.
By way of non-limiting example, the aforementioned binding partners may be antibodies. The label may be colloidal gold. The sample may be a biological sample such as whole blood, serum, plasma, or urine. By way of example, the preselected analyte may be a cardiac analyte, such as myoglobin, and the corresponding marker may be carbonic anhydrase III. For determining the level of myoglobin originating from the heart, the mobile, labeled binding partner to the marker may be a gold-labeled monoclonal anti-carbonic anhydrase III antibody, the conjugate between the marker and streptavidin may be a conjugate between carbonic anhydrase III and streptavidin, the biotinylated binding partner to the preselected analyte may be biotinylated anti-myoglobin monoclonal antibody, and the immobilized binding partner to the preselected analyte may be an immobilized anti-myoglobin monoclonal antibody.
The aforementioned conjugate between the marker and streptavidin may be a covalent conjugate prepared, for example, by use of a homobifunctional or heterobifunctional cross-linking agent or carbodiimide, or may be a single-chain polypeptide on which reside both the marker or an epitope thereof and streptavidin, each retaining its desired activities and ability to participate in the above-mentioned assay. The foregoing example may be used to diagnose a heart attack.
Of course, in the above methods, wherein two binding partners bind to the preselected analyte, each must be capable of recognizing a different binding site on the preselected analyte such that both binding partners can independently bind and permit the final labeled complex to form if the second analyte (marker) is not present at a level relatively greater than that of the first analyte. Moreover, the sensitivities and selectivities of the foregoing assays may be adjusted, for example, depending on the relative levels of the preselected analyte released from the target source, the level released from the non-target source, and the amount of co-release of the non-target-source marker relative to the release of the preselected analyte from the non-target source. The ratios of the components in the various reagents of the assays may be adjusted, and any reduced binding thereby compensated for in another reagent, as an example of the flexibility of the assay for various analytes.
Preferably, the foregoing binding partners are antibodies, and may be monoclonal or polyclonal antibodies. The preselected analyte is preferably a biomolecule, such as a protein, carbohydrate, nucleic acid, lipid, glycoprotein, glycolipid, by way of example, but it is not so limited. The preselected analyte is capable of being recognized by the binding of two different binding partners, preferably antibodies. The preselected analyte may be present in any sample, including that from a human or animal body, foodstuff or food processing or manufacturing facility, domestic or industrial water supply, etc. Preferably, the sample is a bodily fluid from a human. In a preferred embodiment, the sample is whole blood, the preselected analyte is myoglobin, and the marker is carbonic anhydrase III.
The marker that is also present in the sample is preferably a biomolecule, such as a protein, carbohydrate, nucleic acid, lipid, glycoprotein, glycolipid, by way of example, but it is not so limited. The marker is capable of binding to a binding partner for the marker, preferably an antibody, and the presence of any marker in the sample is capable of competing for binding to the binding partner to the marker with a conjugate comprising the marker, as described above.
The multiple steps in the foregoing examples of the assay of the invention described above may be best illustrated by specific example. Elevated levels of circulating myoglobin, a cardiac and skeletal muscle protein, may be diagnostic for a heart attack if the myoglobin is of cardiac (heart) origin and not from skeletal muscle. Elevated circulating myoglobin from skeletal muscle may indicate muscle damage. While other, more specific cardiac markers are available, myoglobin is particularly desirable if its source can be determined, as it is released early following heart muscle damage, in contrast to other more specific cardiac markers, which are detectable later (for example, after six hours). The method of the present invention provides the level of myoglobin of cardiac origin over that of skeletal origin by taking advantage of the simultaneous release from skeletal muscle of carbonic anhydrase III (CAIII) together with myoglobin. Thus, the first method described hereinabove employs a test strip with 1) a mobile, labeled anti-carbonic anhydrase III antibody; 2) a mobile covalent or single-chain polypeptide-containing conjugate between carbonic anhydrase III and anti-myoglobin antibody; and 3) anti-myoglobin antibody immobilized at the capture zone. In the presence of myoglobin in the sample, myoglobin will form an immunocomplex with the mobile carbonic anhydrase III-anti-myoglobin antibody conjugate, which will be captured at the capture zone by the immobilized anti-myoglobin antibody. The mobile, labeled anti-carbonic anhydrase III antibody will bind to the carbonic anhydrase III on the conjugate, forming a positive band due to the presence of the label. However, in the presence of both myoglobin and carbonic anhydrase III in the sample, the level of binding of the labeled anti-carbonic anhydrase antibody to the mobile carbonic anhydrase III-anti-myoglobin antibody conjugate will be reduced and therefore less will be available to bind to the carbonic anhydrase III on the conjugate, and a reduced or negative result will be obtained, depending on the amount of carbonic anhydrase III present in the sample.
In the second method described above which employs slightly different reagents, myoglobin in the sample will form an immunocomplex with the biotinylated anti-myoglobin antibody, which will then be captured at the capture line by the immobilized anti-myoglobin antibody. The conjugate (covalent or single-chain polypeptide) between streptavidin and carbonic anhydrase III will bind to the biotinylated antibody at the capture line, and the mobile, labeled anti-carbonic anhydrase III will further bind to the streptavidin-carbonic anhydrase III conjugate in the complex, forming a positive band. However, in the presence of both myoglobin and carbonic anhydrase III, the level of binding of the mobile, labeled anti-carbonic anhydrase III antibody to the streptavidin-carbonic anhydrase III conjugate will be reduced by the any carbonic anhydrase III in the sample, and therefore will no longer be available to bind to the immobilized complex. A reduced or negative result will be obtained, depending on the amount of carbonic anhydrase III in the sample.
Depending on antibody affinities, sample flow, volumes, and other parameters, the foregoing assay reduces myoglobin detectability approximately to the extent of the presence of carbonic anhydrase III, in a reciprocal relationship. Modifications of the foregoing methods which achieve the same objectives are likewise embraced herein.
It will further be appreciated that formats alternative to the lateral flow, strip-based format can also be utilized to perform the present assay. In particular, and in embodiments of the invention, the assay is performed in ELISA format, using for instance standard microwell trays convenient for use in robotic readers. In this format, for instance, the sample is mixed with the analyte labeling reagent, and the mixture is then contacted with a binding partner for one of the analytes, desirably the preselected analyte that has been immobilized in the standard way. Following incubation to allow formation of the labeling complex, unbound sample is removed by washing, and a reading is then taken of the immobilized label.
Other pairs of first and second analytes for which knowledge of a ratio or difference in levels is diagnostically useful include fatty acid binding protein and carbonic anhydrase III, and myosin light chain and carbonic anhydrase III for the diagnosis of heart attack; and total cholesterol and high-density lipoprotein (HDL) for assessing risk of atherosclerotic diseases.
The invention is also directed to conjugates or single-chain polypeptides comprising a biotin-binding protein or protein fragment, such as streptavidin, and an analyte, or anti-analyte-binding epitope thereof, to provide a reagents useful in the practice of the invention. For example, a single-chain polypeptide comprising streptavidin and carbonic anhydrase III is useful as the reagent which can bind both a biotinylated antibody and an antibody to carbonic anhydrase III, the utility of which in the practice of the invention is evident from the teachings herein. An example is the single-chain polypeptide shown in SEQ ID NO:1, but this is merely illustrative of a wide variety of conjugates of analytes and biotin-binding molecules that are embraced herein, and may be further extended beyond biotin-streptavidin to other high-affinity binding pairs between one molecule and another, each of which may be separately incorporated into reagents and retain their binding activity. The foregoing conjugates preferably may be prepared by recombinant techniques, wherein a single-chain polypeptide comprising the analyte and streptavidin, joined by a linker peptide, are expressed from a single polynucleotide construct. The invention further embraces polynucleotide sequences encoding such conjugates, such as those that encode SEQ ID NO:1. Alternatively, cross-linking agents may be used to form the reagent. Such include homobifunctional, heterobifunctional, carbodiimides, and such conjugation methods involving covalently linking, with or without a spacer, one functional group of a biomolecule to another is well known in the art.
Another reagent embraced by the present invention is a conjugate of single-chain polypeptide comprising a portion of an antibody (or antigen-binding domain thereof) and a biotin-binding protein or fragment thereof, such that the conjugate, single-chain polypeptide or full antibody may independently recognize and bind to both its epitope and to biotin. The use of this reagent in the practice of the present invention will be evident from the teachings herein. By was of non-limiting example, a reagent comprising streptavidin or a biotin-binding portion thereof covalently bound to an anti-carbonic anhydrase antibody, is described. These reagents may be made by, for example, a bifunctional cross-linking reagent to covalently bind the members of the reagent together, or it may be prepared by recombinant methods, for example, in the construction of a polynucleotide that expresses an immunoglobulin heavy chain with a biotin-binding fragment of streptavidin fused, with our without a linker sequence, to the C-terminal portion of the heavy chain. Association of this single-chain hybrid immunoglobulin molecule with the immunoglobulin light chain will provide a modified antibody molecule capable of both recognizing and binding the intended epitope, and also binding to biotin. This is merely illustrative of methods of preparation and is not intended in any way to be limiting.
The invention also embraces polynucleotide sequences encoding such single-chain polypeptide compositions comprising an immunoglobulin light or heavy chain and a biotin-binding protein or peptide such as streptavidin. A non-limiting example is a polynucleotide sequence which encodes SEQ ID NO:1.
Thus, the invention is also directed to a conjugate comprising an analyte or a fragment thereof, and streptavidin or a biotin-binding equivalent thereof e.g., a biotin-binding variant or fragment of streptavidin, wherein independently, the analyte or fragment thereof in the conjugate is capable of being bound by an antibody to the analyte, and said streptavidin or biotin-binding fragment thereof in the conjugate is capable of binding to biotin. In a preferred embodiment, the analyte or fragment thereof is a protein or peptide. In a further embodiment, the protein or peptide analyte or fragment thereof and streptavidin or a biotin-binding fragment thereof reside on a single polypeptide chain. In a non-limiting example, the analyte is carbonic anhydrase III. An example of such a single-chain polypeptide is depicted in SEQ ID NO:1.
In another embodiment, the invention is directed to conjugate comprising (1) an antibody to a first analyte or a binding fragment thereof, and (2) a second analyte (marker) or a fragment or variant thereof which competes with the second analyte for binding to antibody to the second analyte. In conjugated form, the antibody component in the conjugate is capable of binding the first analyte, and the second analyte component is capable of being bound by an antibody to the second analyte. In one embodiment, the second analyte or fragment thereof is a protein or peptide. In a further embodiment, the second analyte or fragment thereof and a heavy chain or light chain of the antibody reside on a single polypeptide chain. In another embodiment, the second analyte is carbonic anhydrase III and the first analyte is myoglobin. In other embodiments, the second analyte is carbonic anhydrase III and the first analyte is fatty acid binding protein, myosin light chain or any other analyte released from cardiac tissue. The invention also embraces polynucleotides encoding a single-chain polypeptide comprising the immunoglobulin light or heavy chain and an analyte or fragment thereof.
The invention is also directed to kits comprising some or all of the various reagents hereinbefore described in order to carry out any of the assays described and variations therefore embraced herein. Referring to the first analyte as analyte and the second analyte as marker, the following kits are embraced herein:
Kit I
1) a labeled binding partner to the analyte;
2) a conjugate between the marker and a second binding partner to the analyte; and
3) an immobilized antibody to the marker.
Kit II
1) a labeled binding partner to the marker
2) a conjugate between the marker and a binding partner to the analyte; and
3) an immobilized second binding partner to the analyte.
Kit III
1) a labeled binding partner to the analyte;
2) either
a) a second binding partner to the analyte conjugated to biotin, and
b) a conjugate between the marker and a biotin-binding reagent or
c) a second binding partner to the analyte conjugated to a biotin-binding reagent, and
d) a conjugate between the marker and biotin; and
3) an immobilized binding partner to the marker.
Kit IV
1) a labeled binding partner to the marker
2) either
a) a binding partner to the analyte conjugated to biotin and
b) a conjugate between the marker and a biotin-binding reagent or
c) a binding partner to the analyte conjugated to a biotin-binding reagent, and
d) a conjugate between the marker and biotin; and
3) an immobilized second binding partner to the analyte.
In the foregoing kits, the binding partners are preferably antibodies or binding portions thereof, and both the binding partner to the analyte and the second binding partner to the analyte capable of simultaneously binding to the analyte. The conjugates comprising the marker may comprise an epitope of the marker. The immobilized binding partner may be provided in the form of a capture line on a test strip, or it may be in the form of a microplate well surface or plastic bead, by way of non-limiting examples. The kits may be used in a homogeneous format, wherein all reagents are added to the sample simultaneously and no washing step is required for a readout, or the kits may be used in a multi-step procedure where successive additions or steps are carried out, with the immobilized reagent added last, with an optional washing step. The teachings herein will allow a skilled artisan to prepare other variations in kit componentry and assay format which carry out the assay of the invention and variations fully embraced herein. Other reagents and instructions may be included with the foregoing reagents.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.